Killing effect of the herpes simplex virus thymidine kinase/ganciclovir enzyme/prodrug system on human nasopharyngeal carcinoma cells

A promising new approach for the gene therapy of cancer is the introduction of the herpes simplex virus thymidine kinase (HSV tk) gene into tumour cells, where the HSV tk gene product converts the nontoxic prodrug ganciclovir (GCV) into its cytotoxic metabolite. We constructed a recombinant plasmid containing the HSV tk gene using standard molecular biology techniques in order to investigate whether the HSV tk/GCV enzyme/prodrug system could kill the human nasopharyngeal carcinoma cell line HNE-1. The recombinant plasmid pcDNA3.1(-) CMV. TK was transfected into the HNE-1 cells by electroporation. The expression of HSV tk by the transfected HNE-1/TK cells was confirmed by mRNA amplification and Western blotting. The growth of HNE-1/TK cells was inhibited by GCV in a dose-dependent manner. The HSV tk/GCV system also demonstrated a considerable bystander effect on co-cultured wild type HNE-1 cells. We conclude that the HSV tk/GCV system could be used as gene therapy for nasopharyngeal carcinoma.     

Contribution of a combination of ponicidin and acyclovir/ganciclovir to the antitumor efficacy of the herpes simplex virus thymidine kinase gene therapy system

We have previously reported that ponicidin (PND), isolated from Rabdosia ternifolia, potentiates the cell-killing activity of antiherpes prodrugs acyclovir (ACV) and ganciclovir (GCV) in human cancer cells expressing herpes simplex virus thymidine kinase (HSV-TK). To extend these in vitro results to in vivo situations, HSV-TK-expressing HeLa cells were injected into nude mice. The in vivo growth of TK(+) HeLa cells was significantly inhibited by coadministration of PND and ACV, or of PND and GCV, compared with single use of ACV or GCV in spite of lower doses of 1 or 0.25 mg/mouse, respectively. These results indicate that there is a good correlation between this in vivo efficacy and previously reported in vitro efficacy. Because of the insufficiency of incorporation of genes into tumors, bystander cell killing has attracted special interest. In the present study, we determined the ability of PND to potentiate the bystander effects of ACV and GCV in both in vitro and in vivo systems. In vitro combined use of PND with ACV or GCV rendered tumor cells more sensitive to the prodrugs, demonstrating a 1.8- to 97-fold or 2.8- to 26-fold reduction in IC(50) compared with ACV or GCV only, respectively, in 1 to 20% of HSV-TK(+) cells. In the in vivo experiments using nude mice injected with 3 or 10% HSV-TK(+) cells, tumor volume was lower in mice treated with a combination of PND and ACV/GCV than in those treated with ACV or GCV only. No toxicity of PND was seen in mice even at a dose 10-fold higher than that used in the in vivo experiments. These novel strategies could provide benefit to ablative cancer gene therapy by making it feasible to use toxic GCV at lower doses and relatively nontoxic ACV.     

Liposomal encapsulation of ganciclovir enhances the efficacy of herpes simplex virus type 1 thymidine kinase suicide gene therapy against hepatic tumors in rats.

Suicide gene therapy based on ganciclovir (GCV) metabolism by transgene herpes simplex thymidine kinase (HSV-1 TK) has been used to selectively kill proliferating cells in clinical settings such as cancer, vascular restenosis, and immunological disorders. We investigated whether encapsulation of ganciclovir (GCV) into liposomes would improve its efficacy, especially against hepatic tumors. Large unilamellar liposomes containing GCV were prepared by reversed-phase evaporation. Pharmacokinetic studies in rats showed that, compared with free GCV, the intravenous injection of liposome-encapsulated GCV (lip-GCV) led to a faster decrease in GCV plasma concentrations, but higher liver-blood ratios. After treatment of syngeneic HSV-1 TK+ liver metastases in rats, histologically active tumors were found in 95% of the transplanted lesions when physiological saline had been given and in 50% when free GCV had been given at 90.2 microM/kg twice daily. This dose is known to be insufficient for the eradication of HSV-1 TK+ tumors. In contrast, only 5% viable tumors were found in rats receiving lip-GCV at this same concentration. Average tumor volumes were 19 +/- 15, 7 +/- 9, and <1 mm3 for the control, free GCV, and lip-GCV groups, respectively. GCV-related toxicity was no longer observed. The results demonstrate that liposomal encapsulation of GCV is feasible and significantly enhances its efficacy against HSV-1 TK+ hepatic tumors.     

Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy

We have previously demonstrated with several cell lines in vitro that hydroxyurea (HU) synergistically enhances ganciclovir (GCV)-mediated cytotoxicity in bystander cells. In this study, we evaluated the role of DNA synthesis inhibition on enhanced bystander killing and assessed whether addition of HU would improve the efficacy of the HSV-TK/GCV system in vivo. Compared with GCV treatment alone, addition of HU resulted in increased DNA synthesis inhibition and delayed progression through S phase following removal of drug. In a xenograft tumor model, 1:10 and 1:1 mixtures of HSVtk- and LacZ-expressing SW620 cells were injected s.c. in the flanks of nude mice and treated i.p. (100 mg/kg GCV, 1500 mg/kg HU) daily for 5 days. Tumors from mice treated with GCV alone grew rapidly and increased to 10 times their initial size in 15.7 +/- 1.8 and 16.0 +/- 0.9 days for 1:10 and 1:1 mixtures, respectively. However, when both GCV and HU were administered in combination, a single complete tumor regression was observed in both the 1:10 and 1:1 groups. In the remaining mice treated with GCV/HU, it took 23.2 +/- 2.1 (1:10) and 26.4 +/- 3.8 days (1:1) to obtain a similar 10-fold increase in tumor size.     

Sustained release of low-dose ganciclovir from a silicone formulation prolonged the survival of rats with gliosarcomas under herpes simplex virus thymidine kinase suicide gene therapy

A silicone formulation of ganciclovir (GCV-pellet) was developed to enhance the cytotoxic effects of herpes simplex virus thymidine kinase suicide gene therapy. The effectiveness of this drug delivery system was assessed in a rat 9L gliosarcoma model. The GCV-pellets (1 mm in length and in diameter) used in this experiment contained a total amount of 0.15 mg of GCV. In vitro experiments demonstrated that GCV was gradually released over a period of 7 days. Five days after stereotactic tumor inoculation into the right caudate nucleus, a herpes simplex virus type 1 (HSV-1) vector expressing herpes simplex virus thymidine kinase (HSV-tk) (T1, 2x10(6) pfu) was administered at the same location. The survival rate of the group treated with the GCV-pellet was compared with that of the T1 group injected intraperitoneally (IP) with GCV (30 mg/kg/day for 7 days). The GCV-pellet-treated group had a significantly prolonged survival (a median of more than 80 days) compared with the GCV IP group (a median of 65 days) and with control groups (P<0.05). The control groups (untreated or receiving only the virus vector) had a survival of 35-38 days. The survival rate of the GCV-pellet group over 80 days was 75%, and all the rats that survived more than 80 days and did not show tumors upon histological examination of the brain were deemed cured. No toxic effects or immunological reactions were observed histologically around the pellet in brain sections from the rats treated with the GCV-pellet. After GCV-pellet inoculation into the tumor, drug concentrations were kept at 1-10 microg/g tissue for 3-4 days. When the same dose of GCV (0.15 mg) in aqueous solution was injected into the tumor, GCV concentrations reached a peak of 0.5 mg/g tissue after 30 min and decreased below measurable level within 12 h. After IP injections of 3 mg GCV, GCV concentrations in the tumor reached a peak of 5.7 microg/g tissue after 30 min and also decreased below measurable level within 12 h. This sustained release of a low and effective GCV dose with the silicone formulation significantly prolonged survival in combinations with HSV-tk expression if compared to IP administration of GCV. Histological examination suggests that the treatment appears to be safe.     

单纯疱疹病毒感染与急性心肌梗死关系的研究

目的 探讨单纯疱疹病毒(HSV)感染与急性心肌梗死之间的关系.方法 采用ELISA方法检测72例急性心肌梗死患者与79例正常对照组血清中HSV-1及HSV-2抗体水平,同时检测两组的IL-6(pg/ml)、TNF-α(pg/ml)、sV-CAM-1(nmol/L)、hsCRP(mg/L)水平;并进行冠心病危险因素调查.结果 急性心肌梗死组HSV-1 IgG抗体阳性率(73.61%)、HSV-2 IgG抗体阳性率(58.33%)显著高于对照组(39.24%,36.71%;P＜0.01,P＜0.05).急性心肌梗死组IL-6(69.08±108.29)、TNF-α(36.79±5.78)、sVCAM-1(30.03±25.86)、hsCRP(23.80±33.44)水平与对照组(22.91±17.37,2.11±3.32,5.53±5.37,2.10±1.79)比较差异有统计学意义(P＜0.01).调整冠心病危险因素前后,HSV-1和HSV-2 IgG抗体阳性率均与急性心肌梗死有相关性(分别HSV-1OR1=4.4,P=0.01;OR2=4.8,P=0.001;OR3=3.5,P=0.012.HSV-2OR1=2.2,P=0.001;OR2=3.5,P=0.006;OR3=3.3,P=0.009).结论 HSV 感染可能通过激发和加重冠状动脉内炎症反应,参与动脉粥样硬化、急性心肌梗死的病理过程.     

Gene transfer to the nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors.

To improve gene transfer to CNS neurons, critical elements of herpes simplex virus 1 (HSV-1) amplicons and recombinant adeno-associated virus (AAV) vectors were combined to construct a hybrid amplicon vector, and then packaged via a helper virus-free system. We tested the HSV/AAV hybrid amplicon vectors for transduction efficiency and stability of transgene expression (green fluorescent protein) in primary neuronal cultures from rat fetal ventral mesencephalon, in comparison with traditional HSV amplicon, AAV, or adenovirus (Ad) vectors at the same multiplicity of infection. The HSA/AAV hybrid vectors transduced the highest number of primary neurons in culture 2 days after infection. As compared with all other vectors tested, only hybrid vectors containing the AAV rep gene maintained the 2-day level of transgene expression over 12 days in culture. This rep-containing hybrid vector was then tested for efficiency and safety in the brain. One month after injection into adult rat striatum (1 x 10(6) transducing units injected), transgene expression was observed within the striatum (ranging from 564 to 8610 cells) and the substantia nigra (via retrograde transport, ranging from 130 to 809 neurons). The HSV/AAV hybrid amplicon vectors transduced predominantly neurons within the striatum, and showed transduction efficacy similar to and in many cases higher than that of HSV amplicon vectors. No immune response was observed in the HSA/AAV hybrid vector-injected brains, as determined by immune markers specific for helper T lymphocytes, cytotoxic T lymphocytes, and microglia. This HSV/AAV hybrid system shows high transduction efficiency and stability in culture. The effective and safe transgene delivery into the nigrostriatal system illustrates its potential for therapeutic application for neurologic disorders, such as Parkinson and Huntington disease.     

Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22.

We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols.     

Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet.

We established a quantitative flow cytometric method for determination of herpes simplex virus type 1 (HSV-1) susceptibility to acyclovir (ACV), ganciclovir, and foscarnet in vitro. Susceptibility was defined in terms of the drug concentration which reduced the number of cells expressing HSV-1 glycoprotein C (gpC) with a fluorescence intensity of > or =10(2) by 50% (IC50). Flow cytometry allowed us to use a high (1.0) as well as a low (0.005) multiplicity of infection, and determination of the IC50 was possible after one or more viral replicative cycles. IC50s were dependent on virus input and on time postinfection. In mixture experiments, 1 to 2% resistant viruses added to a sensitive strain could be detected. The results obtained by flow cytometry showed a good qualitative correlation with those achieved by cytopathic effect inhibitory assay. However, flow cytometry might detect more quantitative differences in drug susceptibility, especially among resistant strains, as confirmed also by determination of intracellular drug phosphorylation. The mean IC50s for ACV-sensitive strains were 0.45 to 1.47 microM, and those for ACV-resistant strains were between 140 and 3,134 microM. Flow cytometric analysis was fast and accurate, automatizable, and highly reproducible. Flow cytometry may be a more powerful tool than standard cytopathic effect-based assays and could have advantages for the detection of low levels of drug resistance or mixtures of sensitive and resistant virus strains.     

Tromantadine: inhibitor of early and late events in herpes simplex virus replication.

Unlike amantadine (1-adamantanamine), tromantadine (N-1-adamantyl-N-[2-(dimethyl amino)ethoxy]acetamide hydrochloride) inhibits herpes simplex virus type 1 (KOS strain)-induced cytopathic effect and virus replication with limited toxicity to the cells. Vero and HEp-2 cells tolerated up to 2 mg of tromantadine per 2 X 10(6) cells for 24-, 48-, or 96-h incubation periods with little change in cell morphology. Treatment of the cells with 10 to 50 micrograms of tromantadine reduced herpes simplex virus-induced cytopathic effect. Treatment with 100 to 500 micrograms of tromantadine inhibited herpes simplex virus-induced cytopathic effect and reduced virus production. Complete inhibition of virus production was observed with treatments of 500 micrograms to 1 mg. The antiherpetic activity of tromantadine was dependent upon the viral inoculum size and the time of addition of the compound with respect to infection. Virion synthesis and viral polypeptide synthesis were inhibited by addition of tromantadine at the time of infection or 4 h postinfection. The results are consistent with tromantadine inhibition of an early event in herpes simplex virus infection, before macromolecular synthesis, and a late event, such as assembly or release of virus.     

人乳头瘤病毒和单纯疱疹病毒与宫颈癌的关系

目的探讨人乳头瘤病毒（HPv）和单纯疱疹病毒（HSV）等对宫颈癌的病因学作用。方法应用聚合酶链反应（PCR）-核酸内切酶分型检测宫颈癌活检组织中HPV—DNA和HSV—DNA基因,以正常宫颈组织作对照。结果在宫颈癌活检组织中HPV-16,18,35型和HSV—Ⅱ型阳性率分别为38．9％争36．2％,与正常妇女宫颈组织阳性率均为3．2％比较,差异均有非常显著意义（P〈0．001）。结论HPV-16,18型和HSV-Ⅱ型是诱发宫颈癌的重要病原因子。     

慢病毒载体介导单纯疱疹病毒胸苷激酶/更昔洛韦系统抑制移植物抗宿主病的实验研究

目的研究慢病毒载体介导单纯疱疹病毒胸苷激酶／更昔洛韦（HSV-TK／GCV）系统对小鼠异基因骨髓移植（allo—BMT）后移植物抗宿主病（GVHD）的防治作用。方法将转染HSV-TK基因的慢病毒感染供鼠（C57BL／6小鼠）脾脏淋巴细胞，将感染后的淋巴细胞与供鼠骨髓细胞混合，移植给经。Co1射线照射后的受鼠（BALB／c小鼠）。分别于移植后当天、移植后7天（＋7天）和＋12天腹腔注射GCV25mg／kg×7d，观察受鼠生存期、GVHD发生率及严重程度、T细胞亚群（CD3、CD4、CD8）及异基因嵌合等指标。结果HSV-TK／GCV使用0天、＋7天、＋12天组受鼠生存时间分别为（30．10±5．21）d、（36．40±5．28）d、（28．20±4．82）d，三组生存时间均较移植对照组[（15．10±0．43）d]明显延长（P〈0．05）；HSV-TK／GCV＋7天组小鼠50d存活率达60％，高于HSV-TK／GCV0天（40％）和＋12天组（30％）（P〉0．05）。对照组小鼠全部发生Ⅲ～Ⅳ级GVHD，实验组死亡小鼠有Ⅱ～Ⅲ级GVHD病理学改变，而长期生存小鼠仅出现Ⅰ～Ⅱ级GVHD。在＋5，＋10，＋15d，3个时间点实验组小鼠CD4^＋细胞明显高于对照组（P〈0．05），CD8^＋细胞均低于对照组（P〈0．05）。＋30天受鼠异基因嵌合率为100％。结论慢病毒载体介导的HSV-TK／GCV系统能有效控制小鼠allo—BMT后GVHD；＋7天外周血白细胞数开始回升时用GCV控制GVHD效果最佳。     

Potent in vivo antiviral activity of the herpes simplex virus primase-helicase inhibitor BAY 57-1293

BAY 57-1293 belongs to a new class of antiviral compounds and inhibits replication of herpes simplex virus (HSV) type 1 and type 2 in the nanomolar range in vitro by abrogating the enzymatic activity of the viral primase-helicase complex. In various rodent models of HSV infection the antiviral activity of BAY 57-1293 in vivo was found to be superior compared to all compounds currently used to treat HSV infections. The compound shows profound antiviral activity in murine and rat lethal challenge models of disseminated herpes, in a murine zosteriform spread model of cutaneous disease, and in a murine ocular herpes model. It is active in parenteral, oral, and topical formulations. BAY 57-1293 continued to demonstrate efficacy when the onset of treatment was initiated after symptoms of herpetic disease were already apparent.     

Liver failure caused by herpes simplex virus thymidine kinase plus ganciclovir therapy is associated with mitochondrial dysfunction and mitochondrial DNA depletion

Herpes simplex virus thymidine kinase (HSV-tk) converts ganciclovir (GCV) into an active compound, which can be incorporated into DNA molecules and terminate DNA synthesis. Gene transfer of HSV-tk followed by GCV administration has been used with success to treat experimental cancer and this strategy has entered into clinical trials. Although it is thought that the cytotoxic effect occurs mainly in tumoral dividing cells, where mitotic activity favors integration of the genotoxic compound into nuclear DNA, there are concerns of potential damage to normal nondividing cells. In the present work we have explored the mechanisms of HSV-tk/GCV toxicity and in particular whether this therapy may cause lesions of mitochondrial DNA (mtDNA) and mitochondrial dysfunction. We found that the administration of GCV to rats injected with adenovirus encoding HSV-tk induced hepatocellular damage characterized by the presence of apoptotic bodies, ballooning of hepatocytes, and severe hepatic steatosis with mitochondria enlargement and cristae dissolution at the ultrastructural level. Remarkably, Southern blot analysis showed substantial reduction in the amount of mtDNA in the liver. Using radiolabeled GCV we could demonstrate incorporation of this compound into both nuclear and mtDNA in HSV-tk-transduced rat hepatocytic cell line MCA-RH7777 and subsequent alteration of mitochondrial function. Our observations confirm that GCV can damage both nuclear and mtDNA in cells transduced with HSV-tk and that this effect could be responsible for severe mitochondrial dysfunction and toxicity in normal nondividing cells. These data are relevant for the design of clinical trials using adenoviral vectors encoding HSV-tk.     

A combined indirect elisa and immunoblotting for the detection of intrathecal herpes simplex virus igg antibody synthesis in patients with herpes simplex virus encephalitis

A combined indirect ELISA and immuno-blotting assay was used for the detection of intrathecal synthesis of IgG antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). By using these two assays as well as three markers for blood-brain barrier, leakage can be easily excluded. A total of 21 sera and 24 cerebrospinal fluid (CSF) samples from 11 patients with HSVE were examined. For seven patients more than one pair of serum and CSF were available. For one patient IgG antibodies began to be detectable in CSF after the sixth day from the onset of the disease. In the other 10 patients the intrathecal synthesis of HSV IgG antibodies was detected later than the sixth day and reached high optical density (OD) values after the 10th day from the onset of disease, at the earliest. In contrast, intrathecal HSV antibody synthesis was not found in specimens taken from 20 patients with acute meningitis who composed our negative control group. The use of a combined indirect ELISA and of an immunoblotting assay on a single dilution of serum and CSF for HSV IgG synthesis in the central nervous system (CNS) allowed the diagnosis of HSVE after the first week of disease.     

单纯疱疹病毒感染与冠心病急性心肌梗死血液流变特性的相关性研究

目的　探讨单纯疱疹病毒 (HSV)感染与冠心病心肌梗死的关系。方法　测定 5 1例急性心肌梗死 (AMI)和 4 2例陈旧性心肌梗死 (OMI)患者及 31例冠脉造影正常者 (NC)的HSV - 1特异性抗体 (HSV - 1IgG、HSV - 1IgM)浓度 ,并同步观察纤维蛋白原 (Fg)、血液流变特性指标变化及其与HSV - 1感染的相关性。结果　AMI组HSV - 1IgG阳性率及平均浓度明显高于NC组 (P <0 0 5 ) ,HSV - 1DNA检测结果与之吻合。校正冠心病危险因素前后HSV - 1IgG阳性与AMI均有相关关系 (OR4 2 6 6 ,P =0 0 19;OR 3 82 1,P =0 0 32 )。AMI组IgG、Fg、血浆黏度、低 /高切全血黏度、红细胞压积、红细胞聚集指数高于 ,而红细胞变形指数低于NC组及OMI组 (P均 <0 0 5 )。AMI组中HSV - 1( )组上述指标 (除HCT外 )的改变与同组中HSV - 1( - )组及与NC、OMI组中HSV - 1( )组比较有显著性差异 (P均 <0 0 5 ) ,AMI组中HSV - 1( )组IgG与Fg、血浆黏度、低 /高切全血黏度、红细胞聚集指数呈正相关 ,而与红细胞变形指数呈负相关 ,调整冠心病的危险因素前后IgG与Fg均呈正相关。结论　HSV - 1感染与CHD(AMI)之间存在明显的相关性 ,与Fg、血液流变特性指标也存在相关性。     

Herpes simplex virus thymidine kinase-mediated suicide gene therapy using nano/microbubbles and ultrasound

A physical method using ultrasound (US) and nano/microbubbles (NBs) can deliver exogenous molecules noninvasively into a specific target site. In this study, we evaluated the application of this technology to cancer gene therapy using prodrug activation therapy. Low-intensity pulsed ultrasound (1 MHz; 1.3 W/cm(2)) and NBs were used to transduce the herpes simplex thymidine kinase (HSVtk) gene in vitro, leading to gene transfer. The addition of ganciclovir (GCV) to the transduced cells led to HSVtk/GCV-dependent cell death mediated by apoptosis. This technology was then assessed in vivo, using mice bearing subcutaneous tumors (1 MHz; 3.0 W/cm(2)). Gene transfer to the tumor, measured by luciferase activity, was transient, with a peak of expression 24 h after transduction, and decreased at 48 h, demonstrating the transient nature of US/NB-mediated gene transfer. The therapeutic potential of this approach was evaluated through repeated intratumoral gene delivery using US/NB-mediated transfer of the HSVtk gene, followed by recurrent administration of GCV, using two different experimental treatment protocols. In both cases, dramatic reductions of the tumor size by a factor of four were observed. Altogether, these data demonstrate the potential of US/NB as a new physical gene delivery method for cancer gene therapy. (E-mail: kodama@tubero.tohoku.ac.jp).     

Unlabeled probes for the detection and typing of herpes simplex virus

BACKGROUND: Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. METHODS: Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. RESULTS: The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. CONCLUSIONS: Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.